The objective of the proposed research is to identify roles for microRNAs and other small RNAs in the etiology of mental retardation and fragile X syndrome (FXS). FXS is usually caused by an expanded CGG trinucleotide repeat in the 5'untranslated region (51 UTR) of the gene encoding the fragile X mental retardation protein (FMRP), FMR1. Expansion of the repeat to >200 copies results in DMA methylation of the FMR1 promoter and loss of FMRP expression. The methylation of FMR1 induced by the expanded CGG repeat may involve aberrant recruitment of the RNA interference (RNAi) pathway since RNAs containing an expanded CGG repeat are substrates of the RNAi ribonuclease, Dicer. CGG repeat-derived small RNAs could contribute to FMR1 gene methylation through a recently-identified RNAi mechanism. The pathogenesis of FXS may also involve RNAi. FMRP associates with the RNA-induced silencing complex (RISC), suggesting a role for the FMRP-RISC interaction. Additionally, FMRP binds mRNAs predicted to bind microRNAs, including the mRNA encoding RhoA, a critical regulator of the cytoskeleton that is required for normal human neurodevelopment. The RhoA 3'UTR contains consensus binding sites for FMRP and microRNAs, and aberrant translational regulation may account for the cytoskeletal phenotype of FMRP deficiency seen in axons and dendrites. This application focuses on FXS: its cause, its pathogenesis, and a FMRP mRNA target, RhoA, whose misregulation may be involved in FXS and other mental retardation disorders. We have found that transcripts for RhoA are localized to developing axons, and that local RhoA translation regulates the axonal cytoskeleton. As part of our overall goal to understand the role of microRNAs and other small RNAs in FXS, the specific aims of this application are (1) to determine if CGG repeat expansion results in FMR1 methylation through the RNA interference pathway. In this aim, we use Dicer-/- cells and Dicer inhibitors to assess the role of RNAi in FMR1 methylation. (2) To determine the role of the microRNA pathway in the regulation of RhoA translation. In this aim, we identify "local" microRNAs and determine their role in the regulation of intra-axonal RhoA translation and cytoskeletal remodeling. (3) To determine the role of the FMRP-Dicer pathway in the regulation of RhoA in developing axons. In this aim, we describe experiments that use FMR1 null mice and mice harboring a floxed Dicer allele to determine the role of FMRP in RhoA regulation and how the microRNA pathway interacts with FMRP to mediate its effects on translation and on the cytoskeleton. The experiments in this application address several emerging areas of RNAi, including RNA-directed gene methylation, the regulation of intra-axonal translation, roles and identities of "local" microRNAs, and the role of RNAi in FMRP signaling. Together, the experiments in this application will advance our understanding of microRNAs in FXS and provide insight into pathways that may be relevant for trinucleotide repeat-associated diseases, mental retardation, and related disorders.